Although the conformational conversion of PrPC into PrPSc is the central molecular event in prion diseases, the biological function of PrPC, a normal cell-surface glycoprotein, remains a mystery. Cell biological work from my laboratory has demonstrated that PrPC constitutively cycles between the plasma membrane and an early endosomal compartment, and that clathrin-coated pits mediate internalization of the protein. The significance of this recycling pathway has been obscure, although it suggested the possibility that the protein might serve as a receptor for uptake of an extracellular ligand. However, the identity of this putative ligand was unknown. Several intriguing new pieces of data from other laboratories now suggest a candidate ligand for PrPC: copper ions. Copper ions bind with low micromolar affinity to the histidine-rich peptide repeats that are found in the N-terminal half of PrPC, and membrane fractions from the brains of PrP0/0 mice that do not express PrPC show a markedly reduced content of ionic copper. This new information on the interaction of copper with PrPC can be melded with our own data on cellular trafficking of the protein to suggest a specific and testable hypothesis about the physiological function of PrPC: We propose that PrPC is a copper receptor that serves to increase the efficiency of copper uptake by cells in the CNS. Specifically, we suggest that, by virtue of the cellular recycling pathway that it follows, PrPC can continuously deliver copper ions from the extracellular milieu to an early endosomal compartment within which the bound ions dissociate and are moved into the cytosol by transmembranetransporters. To test this hypothesis, we propose in AIM number 1 to analyze the effect of copper ions on the endocytic trafficking of wild-type and N- terminally deleted forms of PrPC expressed in transfected neuroblastoma cells. In AIM number 2, we will characterize the uptake of radioactive 64Cu by transfected neuroblastoma cells expressing PrPC, and by cerebellar and cortical neurons cultured from the brains of normal mice, PrP0/0 mice, and transgenic mice over-expressing wild-type or N- terminally deleted PrP. The data to be obtained in the proposed experiments will serve as an important starting point for a number of future studies into the role of copper in the biology of PrP and it associated diseases.